Article Volatile Compositions and Antifungal Activities of Native American Medicinal Plants: Focus on the

Sims K. Lawson 1, Layla G. Sharp 1, Chelsea N. Powers 1, Robert L. McFeeters 1, Prabodh Satyal 2 and William N. Setzer 1,2,* 1 Department of Chemistry, University of in Huntsville, Huntsville, AL 35899, USA; [email protected] (S.K.L.); [email protected] (L.G.S.); [email protected] (C.N.P.); [email protected] (R.L.M.) 2 Aromatic Research Center, 230 N 1200 E, Suite 100, Lehi, UT 84043, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-256-824-6519

 Received: 26 December 2019; Accepted: 16 January 2020; Published: 19 January 2020 

Abstract: In the past, Native Americans of North America had an abundant traditional herbal legacy for treating illnesses, disorders, and wounds. Unfortunately, much of the ethnopharmacological knowledge of North American Indians has been lost due to population destruction and displacement from their native lands by European-based settlers. However, there are some sources of Native American ethnobotany remaining. In this work, we have consulted the ethnobotanical literature for members of the Asteraceae used in Cherokee and other Native American traditional medicines that are native to the southeastern United States. The aerial parts of Eupatorium serotinum, Eurybia macrophylla, Eutrochium purpureum, canadensis, Rudbeckia laciniata, Silphium integrifolium, uvedalia, Solidago altissima, and Xanthium strumarium were collected from wild-growing plants in north Alabama. The plants were hydrodistilled to obtain the essential oils and the chemical compositions of the essential oils were determined by gas chromatography–mass spectrometry. The essential oils were tested for in-vitro antifungal activity against Aspergillus niger, Candida albicans, and Cryptococcus neoformans. The essential oil of E. serotinum showed noteworthy activity against C. neoformans with a minimum inhibitory concentration (MIC) value of 78 µg/mL, which can be attributed to the high concentration of cyclocolorenone in the essential oil.

Keywords: ethnopharmacology; essential oil; chemical composition; Cryptococcus neoformans; cyclocolorenone

1. Introduction Many aspects of modern medicine have relied on the traditional knowledge of native cultures, including, for example, traditional Indian medicine (Ayurveda) [1], traditional Chinese medicine (TCM) [2], and traditional Islamic medicine [3]. Unfortunately, many of the traditional uses of medicinal plants are being lost due to several reasons. Recent generations are less interested in traditional knowledge, and habitat destruction and forced migration have reduced access to medicinal plants. The Native Americans of North America also had rich traditions of medicinal plant use. However, much of this knowledge has been lost due to population declines and displacement from native lands. Nevertheless, there are still some existing references to the ethnobotanical uses of medicinal plants by Native Americans [4]. Eupatorium serotinum Michx., “late boneset”, is native to eastern North America and ranges from , , and , to the Atlantic coast and from the Gulf of north to Wisconsin and Michigan [5]. The Houma people of Louisiana used a decoction of the flowers to treat typhoid fever [6]. Extracts of the aerial parts of E. serotinum have yielded germacranolide sesquiterpenoids [7,8].

Plants 2020, 9, 126; doi:10.3390/plants9010126 www.mdpi.com/journal/plants Plants 2020, 9, 126 2 of 18

Eurybia macrophylla (L.) Cass. (syn. Aster macrophyllus L.), “bigleaf aster”, is native to southeastern Canada and northeastern United States, as far south as north Alabama and north [9]. The Iroquois used a decoction of the roots as a blood medicine and to treat venereal disease; the Ojibwa people ate the leaves of E. macrophylla as a medicine and food [6]. Eutrochium purpureum (L.) E.E. Lamont (syn. Eupatorium purpureum L.), “purple Joe-Pye weed”, ranges from central to eastern North America, from the Great Lakes region south to the Gulf of Mexico [10]. The Cherokee used the roots as a treatment for rheumatism, for kidney problems, and for “female problems”; the Chippewa inhaled the vapors from an infusion of the plant for colds; the Navajo used the plant as an antidote for poison; and the Potawatomi people applied a poultice of the leaves on burns [6]. L., “white flower leafcup”, is found in eastern North America from Alabama and Georgia north to Ontario, and from Kansas and Oklahoma east to the Appalachians and New York [11]. The Houma people applied a poultice of the crushed leaves to swellings; the Iroquois used the plant to relieve toothache [6]. Extracts of the aerial parts of P. canadensis have yielded diterpenoid carboxylic acids and germacranolides [12]. Rudbeckia laciniata L., “cutleaf coneflower”, is widespread in the United States and Canada [13]. There are eight varieties of R. laciniata, namely ampla, bipinnata, digitata, gaspereauensis, heterophylla, hortensia, humilis, and laciniata [14]; R. laciniata var. laciniata is the common variety found in eastern North America [15]. The Cherokee ate the cooked greens to “keep well”, while the Chippewa applied a poultice of the flowers to treat burns [6]. Several lignans, flavonoid glycosides, and quinic acid derivatives have been isolated from the aerial parts of R. laciniata, and sesquiterpenoids have been isolated from root extracts [4]. Silphium integrifolium Michx., “whole-leaf rosinweed”, ranges from Wisconsin and Michigan, south through Alabama and Mississippi, and west as far as New Mexico [16]. An infusion of the leaves of S. integrifolium was taken by the Meskwaki people for “bladder troubles” [6]. Flavonoids, oleanolic acid glycosides, and phenolic acids have been identified in the aerial parts of S. integrifolium [17]. (L.) Mack. (syn. Polymnia uvedalia (L.) L.), “yellow flower leafcup”, is found in the southeastern United States from Virginia to , west to eastern Texas and Oklahoma [18]. The Cherokee used the bruised roots on burns and cuts; the Iroquois took an infusion of the shoots and roots to treat back pain and vomiting [6]. The plant is the source of several germacranolide sesquiterpenoids and ent-kaurane diterpenoids [4]. Solidago altissima L. (syn. Solidago canadensis L.), “Canada goldenrod”, ranges across most of North America from Canada to northern Mexico [19]. The Okanagan-Colville and the Thompson tribes used an infusion of the roots and shoots of S. altissima to treat fevers [6], and the Cherokee took an infusion of Solidago spp. to treat fevers. The phytochemistry of S. canadensis has been extensively studied and found to contain saponins [20,21], flavonoids [22–24], polyacetylenes [25,26], diterpenoids [27], and triterpenoids [28]. Xanthium strumarium L., “rough cocklebur”, ranges throughout North America and is considered a noxious weed in the southeastern United States [29]. The White Mountain Apache tribe took a root decoction to treat fevers; the Mahuma people of Southern California used the plant to treat rheumatism, tuberculosis, and gonorrhea [6]. The aerial parts of X. strumarium contain alkaloids, sesquiterpene lactones (guaianolides, germacranolides, and elemanolides), phenolic compounds, and the toxic carboxylic acid atractyloside, a kaurene glycoside [30]. We have had an interest in the volatile chemistry and biological activity of Native American medicinal plants [31–42], including members of the Asteraceae [43–45]. As part of our continuing investigations, the purpose of this work was to seek out additional species of Asteraceae important in Native American traditional medicine growing wild in northern Alabama and to obtain the essential oils by hydrodistillation of the aerial parts. As a test for biological activity, the essential oils were then screened for antifungal activity against three potentially pathogenic fungal strains. Aspergillus niger, Candida albicans, and Cryptococcus neoformans are the causative agents of opportunistic Aspergillus lung disease, candidiasis, and cryptococcosis, respectively. Plants 2020, 9, 126 3 of 18

2. Results and Discussion The essential oils from E. serotinum, E. macrophylla, E. purpureum, P. canadensis, R. laciniata, S. integrifolium, S. uvedalia, S. altissima, and X. strumarium were obtained from the fresh aerial parts of the plants by hydrodistillation, generally in low yield. The essential oils were analyzed by GC and GC–MS (Tables 1, 3–9, and 11).

2.1. Eupatorium serotinum Michx. The essential oil from the aerial parts of E. serotinum was rich in sesquiterpenoids, with cyclocolorenone (23.38%), germacrene D (6.58%), and palustrol (5.32%), along with an unidentified sesquiterpenoid (5.72%) as the major components (Table1).

Table 1. Chemical composition of the essential oil of Eupatorium serotinum Michx.

RI a RI b Compound % SD RI a RI b Compound % SD ± ± 802 801 Hexanal 0.16 0.02 1531 1533 trans-Cadina-1,4-diene 0.20 0.07 ± ± 810 796 2-Hexanol 0.92 0.01 1540 — Unidentified e 1.28 0.05 ± ± 850 846 (2E)-Hexenal 0.86 0.11 1542 1539 α-Copaen-11-ol 7.89 0.13 ± ± 932 932 α-Pinene 0.17 0.01 1548 — Unidentified f 1.76 0.04 ± ± 949 946 Camphene 1.78 0.02 1550 — Unidentified g 0.75 0.03 ± ± 977 974 β-Pinene 0.18 0.02 1558 1559 Germacrene B 0.44 0.03 ± ± 999 1001 δ-2-Carene 0.15 0.01 1562 — Eudesmenol h 0.32 0.09 ± ± 1029 1024 Limonene 0.26 0.01 1569 1567 Palustrol 5.32 0.12 ± ± 1283 1287 Bornyl acetate 4.72 0.07 1575 1574 Germacra-1(10),5-dien-4β-ol 0.91 1.10 ± ± 1326 — Unidentified c 0.93 0.03 1581 1577 Spathulenol 1.58 0.83 ± ± 1346 1345 α-Cubebene 0.59 0.01 1588 1590 Globulol 0.58 0.03 ± ± 1375 1374 α-Copaene 0.11 0.05 1593 1592 Viridiflorol 1.12 0.09 ± ± 1383 1387 β-Bourbonene 0.06 0.01 1596 — Unidentified i 1.20 0.02 ± ± 1397 1387 β-Cubebene 3.65 0.09 1603 1602 Ledol 2.80 0.03 ± ± 1406 1409 α-Gurjunene 0.74 0.02 1620 1611 Germacra-1(10),5-dien-4α-ol 1.44 0.13 ± ± 1417 1419 β-Ylangene 0.09 0.03 1622 1624 Selina-6-en-4β-ol 0.31 0.03 ± ± 1418 1417 β-Caryophyllene 0.96 0.01 1627 1627 1-epi-Cubenol 0.61 0.10 ± ± 1428 1434 γ-Elemene 0.28 0.05 1638 1639 cis-Guaia-3,9-dien-11-ol 0.12 0.01 ± ± 1448 1448 cis-Muurola-3,5-diene 0.07 0.03 1642 1638 τ-Cadinol 0.80 0.03 ± ± 1455 1452 α-Humulene 0.41 0.04 1642 1640 τ-Muurolol 0.62 0.08 ± ± 1471 1475 trans-Cadina-1(6),4-diene 0.25 0.03 1646 1644 α-Muurolol (=δ-Cadinol) 0.69 0.07 ± ± 1480 1484 Germacrene D 6.58 0.09 1648 1646 Agarospirol II 1.10 0.04 ± ± 1486 1488 δ-Selinene 0.27 0.02 1654 1652 α-Cadinol 2.31 0.04 ± ± 1488 1489 β-Selinene 0.17 0.01 1668 — Unidentifiedj 5.72 0.14 ± ± 1491 1493 trans-Muurola-4(14),5-diene 0.69 0.03 1751 1759 Cyclocolorenone 23.38 0.43 ± ± 1494 1493 epi-Cubebol 1.81 0.03 Green leaf volatiles 1.94 ± 1497 1500 α-Muurolene 0.34 0.02 Monoterpene hydrocarbons 2.53 ± 1502 — Unidentified d 1.30 0.02 Oxygenated monoterpenoids 4.72 ± 1512 1513 γ-Cadinene 0.21 0.00 Sesquiterpene hydrocarbons 19.14 ± 1514 1514 Cubebol 4.18 0.11 Oxygenated sesquiterpenoids 57.90 ± 1517 1522 δ-Cadinene 3.02 0.25 Total Identified 86.23 ± a RI = Retention index determined in reference to a homologous series of n-alkanes on a ZB-5ms column. b RI values from the databases. c MS(EI) (mass spectrum (electron impact)): 162(84%), 147(96%), 133(20%), 120(32%), 119(41%), 108(35%), 105(100%), 91(63%), 79(29%), 77(22%), 55(11%), 53(12%), 41(14%). d MS(EI): 202(7%), 187(9%), 162(68%), 159(31%), 147(50%), 145(32%), 132(49%), 119(66%), 105(89%), 91(48%), 81(18%), 79(20%), 77(16%), 59(100%), 43(20%), 41(20%). e MS(EI): 202(4%), 187(13%), 162(56%), 159(59%), 147(39%), 145(40%), 132(73%), 131(39%), 119(73%), 106(48%), 105(88%), 91(47%), 81(16%), 79(25%), 77(19%), 59(100%), 55(18%), 43(19%), 41(20%). f MS(EI): 220(24%), 205(17%), 163(19%), 120(35%), 110(100%), 105(20%), 95(35%), 69(44%), 55(20%), 41(24%). g MS(EI): 220(47%), 163(25%), 161(32%), 121(100%), 108(42%), 93(42%), 81(59%), 69(17%), 55(15%), 41(18%). h Correct isomer not identified. i MS(EI): 220(49%), 205(7%), 163(33%), 161(28%), 121(100%), 108(40%), 93(35%), 81(80%), 69(20%), 55(17%), 41(19%). j MS(EI): 202(46%), 187(67%), 174(40%), 162(60%), 159(100%), 147(89%), 134(30%), 131(23%), 119(62%), 105(71%), 91(50%), 59(61%), 43(20%), 41(22%).

To our knowledge, there have been no previous reports on the essential oil composition of E. serotinum. The phytochemistry of the Eupatorium has been reviewed [46] and there have been numerous reports on the essential oil compositions from other species of the genus (Table2). There is much variability in the essential oil compositions of Eupatorium species, both between species and within species. Nevertheless, sesquiterpenoids often dominate the essential oils of Eupatorium species. Plants 2020, 9, 126 4 of 18

Table 2. Major components and biological activities of Eupatorium essential oils.

Eupatorium spp. Essential Oil Location Major Components Biological Activity Ref. camphene (8.9%), p-cymene (16.6%), bornyl acetate (15.6%), amorph-4-en-7-ol (9.6%), E. adenophorum (aerial parts) Nainital, India none reported [47] α-cadinol (6.2%), amorpha-4,7(11)-dien-8-one (7.8%) bornyl acetate (9.0%), germacrene D (5.7%), β-bisabolene (6.2%), 1-naphthalenol E. adenophorum (leaves) Palampur, India Antibacterial (Rhodococcus rhodochrous, MBC 12.5 µL/mL) [48] (17.5%), α-bisabolol (9.5%) camphene (12.1%), α-phellandrene (8.6%), α-terpinene (6.5%), p-cymene (11.6%), Antibacterial (Erwinia herbicola, MIC 0.25 µL/mL; E. adenophorum (twigs) Uttar Pradesh, India [49] bornyl acetate (10.6%), acoradiene (10.1%), α-bisabolol (5.3%) Pseudomonas putida, MIC 2.0 µL/mL) Antifungal (Macrophomina phaseolina, EC 0.076 µL/mL; bornyl acetate (6.3%), β-caryophyllene (5.4%), γ-muurolene (11.7%), γ-curcumene 50 E. adenophorum (inflorescence) Palampur, India Rhizoctonia solani, EC 0.094 µL/mL; Fusarium oxysporum, [50] (5.7%), γ-cadinene (18.4%), 3-acetoxyamorpha-4,7(11)-dien-8-one (7.4%) 50 EC50 0.120 µL/mL) β-cubebene (5.7%), β-caryophyllene (12.3%), germacrene D (15.5%), δ-cadinene (5.8%), E. amygdalinum (aerial parts) Amapá, none reported [51] caryophyllene oxide (17.4%) α-pinene (17.0%), β-pinene (6.1%), p-cymene (12.5%), thymyl acetate (9.7%), E. argentinum (leaves) Córdoba, none reported [52] β-caryophyllene (7.2%) α-pinene (13.7%), p-cymene (30.0%), β-ocimene (5.3%), thymyl acetate (12.3%), E. arnottianum (aerial parts) Córdoba, Argentina none reported [53] β-caryophyllene (11.7%) limonene (32.7%), piperitenone (21.2%), trans-dihydrocarvone (10.2%), camphor E. arnottianum (aerial parts) Córdoba, Argentina Antiviral (HSV-1, IC 52.1 µg/mL; DENV-2, IC 38.2 µg/mL) [54] (6.8%), cis-dihydrocarvone (6.7%) 50 50 β-caryophyllene (7.9%), γ-elemene (5.9%), germacrene D (9.8%), cadinene (5.8%), E. arnottii (aerial parts) San Luis, Argentina Insecticidal (Tribolium castaneum, ED 0.15 mg/cm2) [55] spathulenol (10.6%), phytol (8.1%) 50 α-pinene (6.2%), sabinene (6.5%), β-pinene (5.4%), myrcene (7.3%), limonene (15.3%), E. ballotaefolium (aerial parts) Ceará, Brazil none reported [56] (E)-β-ocimene (10.5%), β-caryophyllene (7.5%) β-caryophyllene (36.1%), α-humulene (13.3%), γ-muurolene (20.3%), E. betonicaeforme (leaves) Ceará, Brazil Larvicidal (Aedes aegypti, LC 129 µg/mL) [57] bicyclogermacrene (15.0%) 50 E. buniifolium (aerial parts) Canelones, α-pinene (14.7%), β-elemene (12.2%), germacrene D (11.5%), trans-β-guaiene (6.5%) none reported [58] 2 E. buniifolium (aerial parts) San Luis, Argentina α-pinene (51.0%), sabinene (7.5%), limonene (9.6%), β-caryophyllene (5.2%) Insecticidal (Tribolium castaneum, ED50 0.15 mg/cm ) [55]

E. buniifolium (leaves) Canelones, Uruguay α-pinene (8.2%), germacrene D (11.1%), trans-β-guaiene (7.4%) Varroacide (Varroa destructor, LD99 0.3 mg/mL) [59] E. cannabinum ssp. cannabinum Antibacterial (Staphylococcus aureus, Streptococcus fecalis, Agerola, Italy δ-2-carene (6.5%), germacrene D (33.5%), α-farnesene (12.9%) [60] (aerial parts) Bacillus subtilis, Bacillus cereus, MIC 1.25 mg/mL) E. cannabinum (leaves) Tuscany, Italy thymol methyl ether (7.8%), germacrene D (29.2%), spathulenol (7.3%) none reported [61] E. cannabinum ssp. corsicum Corsica, France α-phellandrene (19.0%), p-cymene (5.2%), germacrene D (28.5%) none reported [62] (aerial parts) E. cannabinum (aerial parts) Mazandaran, Iran α-terpinene (17.8%), thymol methyl ether (5.2%), germacrene D (9.1%) none reported [63] thymol methyl ether (5.7%), neryl acetate (9.4%), germacrene D (11.3%), E. cannabinum (leaves) Vilnius, Lithuania none reported [64] β-bisabolene (6.7%) p-cymene (23.7%), thymol methyl ether (8.9%), β-bisabolene (8.2%), E. capillifolium (aerial parts) Cuba none reported [65] selin-11-en-4α-ol (12.3%) myrcene (15.7%), α-phellandrene (6.5%), thymol methyl ether (36.3%), E. capillifolium (aerial parts) Mississippi, USA Insecticidal (Stephanitis pyrioides, LC 5800 µg/mL) [66] 2,5-dimethoxy-p-cymene (20.8%) 50 Plants 2020, 9, 126 5 of 18

Table 2. Cont.

Eupatorium spp. Essential Oil Location Major Components Biological Activity Ref. spathulenol (15.5%), β-caryophyllene (7.8%), germacrene D (5.5%), E. catarium (aerial parts) Córdoba, Argentina Antiviral (HSV-1, IC 47.9 µg/mL; DENV-2, IC 57.3 µg/mL) [54] bicyclogermacrene (5.1%) 50 50 β-caryophyllene (7.1%), α-humulene (6.6%), germacrene D (16.8%), E. conyzoides (aerial parts) Tocantins, Brazil none reported [51] bicyclogermacrene (7.2%), spathulenol (8.3%)

E. glabratum (leaves) Michoacán, México α-pinene (29.5%), β-pinene (6.3%), α-phellandrene (19.6%) Insecticidal (Sitophilus zeamais, LC50 18.0 µL/mL) [67] α-pinene (13.4%), β-pinene (7.8%), β-ocimene (6.2%), carvacrol (7.1%), thymyl acetate E. hecatanthum (leaves) Córdoba, Argentina none reported [52] (10.6%), β-caryophyllene (8.1%) limonene (9.7%), δ-elemene (10.6%), β-caryophyllene (27.7%), α-humulene (5.9%), E. inulaefolium (aerial parts) San Luis, Argentina Insecticidal (Tribolium castaneum, ED 0.15 mg/cm2) [55] patchoulene (9.2%), germacrene D (13.7%), viridiflorol (9.2%) 50 germacrene D (8.6%), selina-3,7(11)-diene (6.1%), spathulenol (5.4%), globulol (16.2%), E. laevigatum (aerial parts) Roraima, Brazil none reported [51] laevigatin (23.6%) E. laevigatum (leaves) Rio Grande do Sul, Brazil germacrene D (11.7%), bicyclogermacrene (9.3%), laevigatin (59.6%) none reported [68] E. macrophyllum (aerial parts) Chapada dos Guimarães, Brazil sabinene (46.7%), limonene (23.3%) none reported [51] ar-curcumene (6.8%), α-zingiberene (57.5%), β-sesquiphellandrene (7.1%), E. marginatum (aerial parts Ananindeua, Pará, Brazil none reported [51] (E)-γ-bisabolene (9.7%) E. marginatum (aerial parts Roraima, Brazil α-gurjunene (19.5%), germacrene D (14.8%), α-selinene (9.0%), (E)-γ-bisabolene (5.0%) none reported [51] α-pinene (8.4%), β-pinene (5.6%), pregeijerene (17.6%), germacrene D (11.1%), E. odoratum (aerial parts) Thitsanulok, Thailand none reported [69] β-caryophyllene (7.3%), vestitenone (6.5%) α-pinene (42.2%), β-pinene (10.6%), β-caryophyllene (5.4%), germacrene D (9.7%), Antibacterial (Bacillus cereus, MIC 39 µg/mL), antifungal E. odoratum (leaves) Lagos, Nigeria [70] β-copaen-4α-ol (9.4%) (Aspergillus niger, MIC 78 µg/mL) E. odoratum (aerial parts) Western Ghats, India cis-sabinene hydrate (5.7%), pregeijerene (14.2%), epi-cubebol (9.8%), cubebol (8.6%) none reported [71] β-caryophyllene (6.2%), germacrene D (21.6%), bicyclogermacrene (6.0%), spathulenol E. squalidum (aerial parts) Amapá, Brazil none reported [51] (14.2%), globulol (25.1%) limonene (6.6%), β-caryophyllene (9.6%), germacrene D (10.4%), E. squalidum (aerial parts) Tocantins, Brazil none reported [51] caryophyllene oxide (30.1%) α-pinene (11.0%), β-pinene (5.9%), p-cymene (24.8%), α-copaene (5.1%), E. subhastatum (leaves) Córdoba, Argentina none reported [52] α-humulene (5.1%) E. triplinerve (leaves) Lucknow, India δ-elemene (5.9%), β-caryophyllene (14.7%), selina-4(15),7(11)-dien-8-one none reported [72] 2 E. viscidum (aerial parts) San Luis, Argentina 6-methyl-5-hepten-2-one (18.2%), spathulenol (25.2%) Insecticidal (Tribolium castaneum, ED50 > 0.212 mg/cm ) [55] Plants 2020, 9, 126 6 of 18

2.2. Eurybia macrophylla (L.) Cass. Monoterpene hydrocarbons, limonene (28.66%), β-pinene (8.57%), and terpinolene (5.35%), and germacrane sesquiterpenes, germacrene D (19.81%), and germacrene B (7.07%), were the major components in the essential oil of E. macrophylla (Table3). To our knowledge, there are no reports on essential oil compositions of any Eurybia species.

Table 3. Chemical composition of the essential oil of Eurybia macrophylla (L.) Cass.

RI a RI b Compound % SD RI a RI b Compound % SD ± ± 801 797 (3Z)-Hexenal 0.06 0.01 1387 1389 β-Elemene 1.48 0.04 ± ± 802 801 Hexanal 0.31 0.05 1418 1417 β-Caryophyllene 4.60 0.07 ± ± 850 846 (2E)-Hexenal 1.44 0.06 1427 1434 γ-Elemene 3.16 0.01 ± ± 865 863 1-Hexanol 0.11 0.02 1431 1432 trans-α-Bergamotene 0.05 0.02 ± ± 924 924 α-Thujene 0.16 0.01 1454 1452 α-Humulene 0.64 0.01 ± ± 932 974 α-Pinene 3.12 0.04 1473 1471 Massoia lactone 0.35 0.04 ± ± 948 946 Camphene 0.60 0.00 1480 1484 Germacrene D 19.81 0.20 ± ± 971 969 Sabinene 0.15 0.02 1487 1489 β-Selinene 0.31 0.05 ± ± 977 974 β-Pinene 8.57 0.07 1494 1500 Bicyclogermacrene 1.95 0.02 ± ± 988 988 Myrcene 1.79 0.02 1497 1500 α-Muurolene 0.16 0.03 ± ± 989 988 Dehydro-1,8-cineole 0.28 0.01 1516 1522 δ-Cadinene 0.19 0.02 ± ± 1006 1002 α-Phellandrene 0.88 0.01 1536 1537 α-Cadinene 0.10 0.02 ± ± 1016 1014 α-Terpinene 0.16 0.02 1557 1559 Germacrene B 7.07 0.07 ± ± 1024 1020 p-Cymene 0.15 0.01 1575 1577 Spathulenol 0.10 0.01 ± ± 1028 1024 Limonene 28.66 0.33 1581 1582 Caryophyllene oxide 0.38 0.01 ± ± 1030 1025 β-Phellandrene 0.75 0.03 1595 1592 Viridiflorol 0.23 0.03 ± ± 1034 1032 (Z)-β-Ocimene 0.18 0.00 1627 1629 iso-Spathulenol 0.08 0.01 ± ± 1044 1044 (E)-β-Ocimene 2.14 0.02 1641 1638 τ-Cadinol 0.09 0.02 ± ± 1057 1054 γ-Terpinene 0.37 0.01 1643 1640 τ-Murrolol 0.12 0.03 ± ± 1084 1086 Terpinolene 5.35 0.08 1646 1644 α-Muurolol (=δ-Cadinol) 0.10 0.01 ± ± 1112 1114 (E)-4,8-Dimethylnona-1,3,7-triene 0.35 0.01 1654 1652 α-Cadinol 0.47 0.02 ± ± 1124 1118 cis-p-Menth-2-en-1-ol 0.72 0.01 1832 1835 Neophytadiene 0.05 0.02 ± ± 1142 1136 trans-p-Menth-2-en-1-ol 0.44 0.01 1838 1841 Phytone 0.05 0.02 ± ± 1187 1179 p-Cymen-8-ol 0.21 0.03 Green leaf volatiles 1.91 ± 1195 1186 α-Terpineol 0.06 0.02 Monoterpene hydrocarbons 53.03 ± 1197 1195 cis-Piperitol 0.15 0.02 Oxygenated monoterpenoids 2.30 ± 1209 1207 trans-Piperitol 0.17 0.01 Sesquiterpene hydrocarbons 40.03 ± 1283 1287 Bornyl acetate 0.27 0.10 Oxygenated sesquiterpenoids 1.59 ± 1292 1293 Undecan-2-one 0.05 0.01 Diterpenoids 0.11 ± 1333 1335 δ-Elemene 0.50 0.00 Others 0.75 ± Total Identified 99.72 a RI = Retention index determined in reference to a homologous series of n-alkanes on a ZB-5ms column. b RI values from the databases.

2.3. Eutrochium purpureum (L.) E.E. Lamont (syn. Eupatorium purpureum L.) The major components in the essential oil of E. purpureum were the green leaf volatiles (2E)-hexenal (60.59%) and hexanal (6.78%), along with the aromatic compounds eugenol (11.68%) and methyl salicylate (10.31%; Table4). There have apparently been no previous reports on the essential oil composition of E. purpureum or any other Eutrochium species. There are numerous reports on Eupatorium essential oils, however (see above).

Table 4. Chemical composition of the essential oil of Eutrochium purpureum (L.) E.E. Lamont.

RI a RI b Compound % SD RI a RI b Compound % SD ± ± 801 797 (3Z)-Hexenal 1.01 0.11 1206 1201 Decanal 0.37 0.05 ± ± 802 801 Hexanal 6.78 0.17 1351 1356 Eugenol 11.68 0.14 ± ± 850 946 (2E)-Hexenal 60.59 1.00 1417 1417 β-Caryophyllene 0.24 0.02 ± ± 865 963 1-Hexanol 2.35 0.41 1479 1484 Germacrene D 0.67 0.10 ± ± 931 932 α-Pinene 1.48 0.09 1559 1561 (E)-Nerolidol 0.50 0.01 ± ± 943 — Unidentified c 0.56 0.07 Green leaf volatiles 71.47 ± 1004 998 Octanal 0.33 0.04 Monoterpene hydrocarbons 2.36 ± 1005 1004 (3Z)-Hexenyl acetate 0.72 0.12 Sesquiterpene hydrocarbons 0.91 ± 1028 1024 Limonene 0.88 0.07 Oxygenated sesquiterpenoids 0.50 ± 1045 1036 Benzene acetaldehyde 0.60 0.03 Benzenoids 22.59 ± 1105 1100 Nonanal 0.91 0.19 Fatty aldehydes 1.61 ± 1192 1190 Methyl salicylate 10.31 0.18 Total Identified 99.44 ± a RI = Retention index determined in reference to a homologous series of n-alkanes on a ZB-5ms column. b RI values from the databases. c MS(EI): 208(8%), 97(100%), 96(17%), 86(9%), 69(12%), 56(22%), 55(64%), 43(18%). Plants 2020, 9, 126 7 of 18

2.4. Polymnia canadensis L. α-Phellandrene (28.30%), α-pinene (19.71%), and germacrene D (11.42%) were the major components in the essential oil from the aerial parts of P. canadensis (Table5). The volatile chemical profile of P. canadensis in this current work is in marked contrast to our previous report on this species [45]. Previous samples were rich in the sesquiterpene hydrocarbons germacrene D (63.6% and 44.5%) and β-caryophyllene (15.9% and 14.8%). The differences in compositions are likely due to seasonal variation (the current sample was collected in July, 2018, while the previous samples were collected in September, 2015, and December, 2016, respectively). We cannot rule out, however, chemical profile differences attributable to environmental differences or biotic differences (e.g., genetics, herbivory, or pathogen stress).

Table 5. Chemical composition of the essential oil of Polymnia canadensis L.

RI a RI b Compound % SD RI a RI b Compound % SD ± ± 802 801 Hexanal 0.28 0.04 1417 1417 β-Caryophyllene 3.05 0.02 ± ± 811 796 2-Hexanol 0.17 0.01 1428 1430 β-Copaene 0.09 0.02 ± ± 850 850 (3Z)-Hexenol 4.31 0.16 1446 1453 Geranyl acetone 0.17 0.01 ± ± 861 854 (2E)-Hexenol 0.08 0.01 1454 1452 α-Humulene 1.14 0.00 ± ± 864 863 1-Hexanol 0.30 0.02 1458 1458 allo-Aromadendrene 0.17 0.02 ± ± 921 921 Tricyclene 0.07 0.01 1479 1484 Germacrene D 11.42 0.01 ± ± 924 924 α-Thujene 0.06 0.01 1484 1486 Phenylethyl 2-methylbutanoate 0.18 0.04 ± ± 932 932 α-Pinene 19.71 0.11 1487 1489 β-Selinene 0.41 0.03 ± ± 948 946 Camphene 0.80 0.01 1490 1490 Phenylethyl 3-methylbutanoate 0.09 0.01 ± ± 971 969 Sabinene 1.96 0.00 1493 1500 Bicyclogermacrene 1.03 0.00 ± ± 976 974 β-Pinene 0.87 0.01 1496 1500 α-Muurolene 0.12 0.01 ± ± 987 988 Myrcene 0.53 0.01 1502 1509 Lavandulyl 3-methylbutanoate 0.76 0.01 ± ± 1006 1002 α-Phellandrene 28.30 0.16 1511 1513 γ-Cadinene 0.21 0.01 ± ± 1016 1014 α-Terpinene 0.09 0.01 1515 1518 Bornyl 3-methylbutanoate 0.39 0.01 ± ± 1024 1020 p-Cymene 4.42 0.02 1516 1522 δ-Cadinene 0.36 0.01 ± ± 1028 1024 Limonene 0.38 0.01 1527 1529 Kessane 0.59 0.04 ± ± 1030 1025 β-Phellandrene 0.06 0.02 1535 1534 Liguloxide 0.84 0.01 ± ± 1034 1032 (Z)-β-Ocimene 0.17 0.01 1559 1561 (E)-Nerolidol 1.71 0.01 ± ± 1044 1044 (E)-β-Ocimene 0.19 0.01 1565 1565 Thymyl 2-methylbutanoate 0.69 0.01 ± ± 1057 1054 γ-Terpinene 0.09 0.01 1568 1570 Neryl 2-methylbutanoate 0.76 0.01 ± ± 1069 1065 cis-Sabinene hydrate 0.08 0.01 1575 1574 Germacrene D-4β-ol 0.18 0.01 ± ± 1084 1086 Terpinolene 0.07 0.01 1581 1582 Caryophyllene oxide 0.21 0.02 ± ± 1099 1095 Linalool tr c 1608 1613 Copaborneol 0.18 0.05 ± 1101 1098 trans-Sabinene hydrate tr 1641 1638 τ-Cadinol 0.59 0.02 ± 1141 1135 trans-Pinocarveol 0.08 0.02 1654 1652 α-Cadinol 0.81 0.02 ± ± 1145 1140 trans-Verbenol 0.14 0.01 1657 1658 Selin-11-en-4α-ol 0.15 0.01 ± ± 1163 1165 Lavandulol 0.12 0.01 1684 1685 Germacra-4(15),5,10(14)-trien-1α-ol 0.49 0.03 ± ± 1172 1165 Borneol 0.11 0.01 1693 1695 6-epi-Shyobunol 0.20 0.02 ± ± 1180 1174 Terpinen-4-ol 0.26 0.00 2227 d Kauran-16β-ol 3.48 0.01 ± ± 1208 1204 Verbenone 0.06 0.01 2243 d Kauran-16α-ol 0.17 0.02 ± ± 1228 1232 Thymol methyl ether 2.89 0.01 Green leaf volatiles 5.13 ± 1342 1345 7-epi-Silphiperfol-5-ene 0.59 0.02 Monoterpene hydrocarbons 57.78 ± 1351 1356 Eugenol 0.18 0.03 Oxygenated monoterpenoids 6.34 ± 1367 1369 Cyclosativene 0.08 0.01 Sesquiterpene hydrocarbons 18.92 ± 1367 1371 Longicyclene tr Oxygenated sesquiterpenoids 5.96 1372 1377 Silphiperol-6-ene 0.06 0.00 Diterpenoids 3.65 ± 1374 1374 α-Copaene 0.12 0.01 Benzenoids 0.45 ± 1380 1382 Modheph-2-ene 0.11 0.00 Others 0.17 ± 1386 1387 β-Cubebene 0.06 0.01 Total Identified 98.40 ± 1387 1389 β-Elemene 0.50 0.01 ± a RI = Retention index determined in reference to a homologous series of n-alkanes on a ZB-5ms column. b RI values from the databases. c tr = “trace” (<0.05%). d Assignment tentative; based on MS only.

2.5. Rudbeckia laciniata L. Monoterpene hydrocarbons dominated the essential oil of R. laciniata (Table6). The major components were limonene (58.07%), α-pinene (10.18%), β-pinene (9.21%), and myrcene (5.26%). While R. laciniata essential oil was rich in monoterpene hydrocarbons, the essential oils of R. fulgida and R. hirta were rich in sesquiterpene hydrocarbons [43]. The major components in R. fulgida essential oil were germacrene D (30.1%), δ-cadinene (17.8%), β-caryophyllene (10.0%), and γ-muurolene (8.9%), along with (E)-β-ocimene (6.2%) and (2E)-hexenal (6.0%). Similarly, the major components of R. hirta essential oil were germacrene D (23.6%), δ-cadinene (16.2%), β-caryophyllene (4.7%), γ-muurolene (8.1%), as well as (E)-β-ocimene (15.2%) and (2E)-hexenal (20.2%) [43]. The leaf essential oil of Rudbeckia triloba, Plants 2020, 9, 126 8 of 18 collected in Bucharest, Romania, was rich in monoterpene hydrocarbons, α-pinene (46.0%), sabinene (9.6%), and β-phellandrene (24.6%), along with germacrene D (6.1%), but devoid of limonene [73]. Thus, there do not seem to be any consistent chemical markers for the Rudbeckia genus.

Table 6. Chemical composition of the essential oil of Rudbeckia laciniata L.

RI a RI b Compound % SD RI a RI b Compound % SD ± ± 802 801 Hexanal 0.05 0.00 1206 1204 Verbenone 0.10 0.03 ± ± 810 796 2-Hexanol 0.34 0.01 1218 1215 trans-Carveol 0.25 0.06 ± ± 922 921 Tricyclene 0.10 0.00 1232 1226 cis-Carveol 0.07 0.02 ± ± 924 924 α-Thujene 0.10 0.01 1243 1239 Carvone 0.49 0.02 ± ± 932 932 α-Pinene 10.18 0.06 1283 1287 Bornyl acetate 2.68 0.02 ± ± 948 946 Camphene 2.24 0.02 1349 1350 α-Longipinene 0.08 0.02 ± ± 971 969 Sabinene 0.90 0.01 1368 1369 Cyclosativene 0.06 0.02 ± ± 977 974 β-Pinene 9.21 0.05 1375 1374 α-Copaene 0.16 0.01 ± ± 988 988 Myrcene 5.26 0.02 1391 1390 Sativene 0.05 0.01 ± ± 1004 1003 p-Mentha-1(7),8-diene 0.07 0.01 1417 1419 β-Ylangene tr ± 1024 1020 p-Cymene 0.11 0.01 1418 1417 β-Caryophyllene 0.43 0.03 ± ± 1029 1024 Limonene 58.07 0.47 1428 1434 γ-Elemene 0.07 0.00 ± ± 1030 1025 β-Phellandrene 0.74 0.08 1431 1432 trans-α-Bergamotene 0.19 0.02 ± ± 1034 1032 (Z)-β-Ocimene 0.09 0.01 1454 1452 α-Humulene 0.12 0.01 ± ± 1044 1044 (E)-β-Ocimene 1.07 0.03 1473 1478 γ-Muurolene 0.05 0.01 ± ± 1069 1067 cis-Linalool oxide (furanoid) 0.19 0.00 1480 1484 Germacrene D 2.52 0.02 ± ± 1085 1084 trans-Linalool oxide (furanoid) 0.05 0.01 1482 1484 (Z,Z)-α-Farnesene 0.05 0.01 ± ± 1121 1119 trans-p-Mentha-2,8-dien-1-ol 0.37 0.01 1494 1500 Bicyclogermacrene 0.06 0.01 ± ± 1130 1131 Limona ketone 0.06 0.01 1497 1500 α-Muurolene 0.07 0.01 ± ± 1132 1132 cis-Limonene oxide 0.20 0.00 1514 1514 Cubebol 0.12 0.01 ± ± 1136 1133 cis-p-Mentha-2,8-dien-1-ol 0.26 0.01 1517 1522 δ-Cadinene 0.15 0.01 ± ± 1136 1137 trans-Limonene oxide 0.27 0.01 1575 1574 Germacra-1(10),5-dien-4β-ol 0.20 0.02 ± ± 1138 1135 Nopinone 0.06 0.01 1581 1582 Caryophyllene oxide 0.19 0.03 ± ± 1140 1135 trans-Pinocarveol 0.19 0.03 1591 1594 Salvial-4(14)-en-1-one tr ± 1145 1140 trans-Verbenol 0.07 0.01 1601 1594 Carotol 0.18 0.01 ± ± 1162 1160 Pinocarvone 0.12 0.00 1620 1611 Germacra-1(10),5-dien-4α-ol 0.20 0.01 ± ± 1171 1165 Borneol 0.11 0.02 Green leaf volatiles 0.39 ± 1178 1179 2-Isopropenyl-5-methyl-4-hexenal 0.08 0.01 Monoterpene hydrocarbons 88.15 ± 1180 1174 Terpinen-4-ol 0.11 0.02 Oxygenated monoterpenoids 6.18 ± 1187 1183 Cryptone 0.10 0.01 Sesquiterpene hydrocarbons 4.06 ± 1195 1195 Myrtenal 0.23 0.02 Oxygenated sesquiterpenoids 0.89 ± 1197 1200 trans-Dihydrocarvone tr c Total Identified 99.67 1199 1195 cis-Piperitol 0.11 0.07 ± a RI = Retention index determined in reference to a homologous series of n-alkanes on a ZB-5ms column. b RI values from the databases. c tr = “trace” (<0.05%).

2.6. Silphium integrifolium Michx. The major components in the essential oil from the aerial parts of S. integrifolium were α-pinene (58.59%) and β-pinene (14.69%), followed by myrcene (9.70%; Table7). Kowalski has extensively examined the essential oils of S. integrifolium as well as S. trifoliatum cultivated in Poland [74–78]. The leaf essential oil of S. integrifolium from Poland had α-pinene (7.3–9.8%), germacrene D (4.0–28.4%), allo-aromadendrene (3.7–8.5%), caryophyllene oxide (6.1–12.4%), and silphiperfol-6-en-5-one (3.7–5.1%) [74,75]; while the floral essential oil was made up of α-pinene (13.4–14.0%), camphene (5.3–5.7%), trans-verbenol (5.2–6.3%), bornyl acetate (6.5–7.0%), and allo-aromadendrene (5.6–6.1%) [74,77]. Thus, there are major qualitative and quantitative differences between the samples from Alabama and from Poland.

2.7. Smallanthus uvedalia (L.) Mack. Monoterpene hydrocarbons dominated the essential oil of S. uvedalia (Table8). α-Pinene (62.56%) was the major component, followed by limonene (11.43%) and β-pinene (6.00%). The chemical composition of this monoterpene-rich essential oil is very different from the compositions collected previously by us [45]. The previous samples, collected in February 2016, were dominated by β-caryophyllene (24.5% and 16.5%) and caryophyllene oxide (19.8% and 14.2%). The sample of S. uvedalia in this present work was collected in September 2018. The differences in composition may be due to seasonal variation, genetic differences, or environmental stresses. Nevertheless, α-pinene has dominated the essential oil compositions of other Smallanthus species. For example, α-pinene was the major component in the essential oil of S. maculatus from (32.9% α-pinene), which was also rich in camphene (5.4%), Plants 2020, 9, 126 9 of 18

β-pinene (7.1%), β-caryophyllene (10.7%), germacrene D (13.7%), and bicyclogermacrene (6.6%) [79]. Likewise, the essential oil of S. quichensis from Costa Rica was also dominated by α-pinene (35.5–64.5%) with lesser concentrations of α-phellandrene (0.1–9.0%), p-cymene (0.1–11.5%), limonene (2.1–5.8%), β-phellandrene (up to 9.2%), and 1,8-cineole (up to 9.7%) [80]. In contrast, S. sonchifolia essential oil, grown in Sichuan, China, was made up of β-phellandrene (26.3%), β-bourbonene (10.2%), β-caryophyllene (14.0%), and β-cubebene (17.6%) [81].

Table 7. Chemical composition of the essential oil of Silphium integrifolium Michx.

RI a RI b Compound % SD RI a RI b Compound % SD ± ± 800 797 (3Z)-Hexenal tr c 1387 1387 β-Cubebene tr 801 801 Hexanal 0.07 0.02 1388 1389 β-Elemene 0.06 0.01 ± ± 810 796 2-Hexanol tr 1417 1419 β-Ylangene tr 849 846 (2E)-Hexenal 0.33 0.02 1418 1417 β-Caryophyllene 2.50 0.02 ± ± 850 844 (3E)-Hexenol 0.27 0.04 1429 1430 β-Copaene tr ± 922 921 Tricyclene 0.12 0.00 1432 1432 trans-α-Bergamotene 0.13 0.02 ± ± 925 924 α-Thujene 0.19 0.00 1454 1452 α-Humulene 1.07 0.02 ± ± 933 932 α-Pinene 58.59 0.21 1469 1471 4,5-di-epi-Aristolochene 0.05 0.00 ± ± 947 945 α-Fenchene tr 1473 1478 γ-Muurolene 0.06 0.01 ± 949 946 Camphene 2.44 0.02 1480 1484 Germacrene D 2.95 0.01 ± ± 971 969 Sabinene 1.78 0.00 1482 1484 (Z,Z)-α-Farnesene 0.10 0.01 ± ± 977 974 β-Pinene 14.69 0.07 1488 1489 β-Selinene 0.15 0.01 ± ± 988 988 Myrcene 9.70 0.02 1491 1493 trans-Muurola-4(14),5-diene tr ± 1004 1003 p-Mentha-1(7),8-diene tr 1494 1500 Bicyclogermacrene 0.08 0.00 ± 1024 1020 p-Cymene tr 1497 1500 α-Muurolene tr 1028 1024 Limonene 1.76 0.01 1512 1513 γ-Cadinene tr ± 1030 1025 β-Phellandrene 0.31 0.03 1517 1522 δ-Cadinene 0.10 0.02 ± ± 1034 1032 (Z)-β-Ocimene 0.05 0.01 1575 1574 Germacra-1(10),5-dien-4β-ol 0.27 0.02 ± ± 1044 1044 (E)-β-Ocimene 0.44 0.03 1581 1582 Caryophyllene oxide 0.47 0.01 ± ± 1057 1054 γ-Terpinene tr 1609 1608 Humulene epoxide II 0.13 0.01 ± 1085 1086 Terpinolene tr 2019 2026 (E,E)-Geranyl linalool 0.06 0.01 ± 1099 1099 α-Pinene oxide 0.10 0.01 2228 2237 7α-Hydroxymanool 0.16 0.02 ± ± 1112 1113 (E)-4,8-Dimethylnona-1,3,7-triene tr 2300 2300 Tricosane tr 1126 1122 α-Campholenal tr 2500 2500 Pentacosane 0.16 0.01 ± 1140 1135 trans-Pinocarveol tr 2700 2700 Heptacosane 0.16 0.02 ± 1145 1140 trans-Verbenol 0.11 0.02 Green leaf volatiles 0.66 ± 1162 1160 Pinocarvone tr Monoterpene hydrocarbons 90.09 1180 1174 Terpinen-4-ol tr Oxygenated monoterpenoids 0.38 1195 1195 Myrtenal 0.06 0.01 Sesquiterpene hydrocarbons 7.37 ± 1206 1204 Verbenone 0.10 0.02 Oxygenated sesquiterpenoids 1.09 ± 1368 1369 Cyclosativene 0.06 0.00 Others 0.32 ± 1375 1374 α-Copaene 0.08 0.01 Total Identified 99.90 ± 1383 1387 β-Bourbonene tr a RI = Retention index determined in reference to a homologous series of n-alkanes on a ZB-5ms column. b RI values from the databases. c tr = “trace” (<0.05%).

Table 8. Chemical composition of the essential oil of Smallanthus uvedalia (L.) Mack.

RI a RI b Compound % SD RI a RI b Compound % SD ± ± 795 801 2-Methylhept-2-ene 0.10 0.00 1207 1204 Verbenone 0.09 0.01 ± ± 801 801 Hexanal 0.86 0.16 1346 1345 α-Cubebene 0.15 0.04 ± ± 850 846 (2E)-Hexenal 1.40 0.09 1382 1387 β-Bourbonene 0.15 0.02 ± ± 865 863 1-Hexanol 0.22 0.01 1418 1417 β-Caryophyllene 3.80 0.07 ± ± 922 921 Tricyclene 0.08 0.00 1454 1452 α-Humulene 0.36 0.02 ± ± 924 924 α-Thujene 1.28 0.02 1473 1478 γ-Muurolene 0.56 0.12 ± ± 932 932 α-Pinene 62.56 0.79 1512 1513 γ-Cadinene 0.29 0.09 ± ± 948 946 Camphene 1.35 0.01 1517 1522 δ-Cadinene 0.63 0.03 ± ± 971 969 Sabinene 0.16 0.03 1536 1537 α-Cadinene 0.22 0.04 ± ± 977 974 β-Pinene 6.00 0.09 1576 1577 Spathulenol 0.58 0.11 ± ± 988 988 Myrcene 2.43 0.07 1581 1582 Caryophyllene oxide 1.37 0.02 ± ± 1024 1020 p-Cymene 0.15 0.01 Green leaf volatiles 2.48 ± 1028 1024 Limonene 11.43 0.11 Monoterpene hydrocarbons 88.28 ± 1030 1025 β-Phellandrene 0.70 0.10 Oxygenated monoterpenoids 0.74 ± 1044 1044 (E)-β-Ocimene 1.87 0.09 Sesquiterpene hydrocarbons 6.16 ± 1057 1054 γ-Terpinene 0.26 0.01 Oxygenated sesquiterpenoids 1.95 ± 1126 1122 α-Campholenal 0.29 0.01 Others 0.10 ± 1140 1135 trans-Pinocarveol 0.23 0.04 Total Identified 99.71 ± 1145 1140 trans-Verbenol 0.14 0.05 ± a RI = Retention index determined in reference to a homologous series of n-alkanes on a ZB-5ms column. b RI values from the databases. Plants 2020, 9, 126 10 of 18

2.8. Solidago altissima L. (syn. Solidago canadensis L.) The major components in the essential oil from the aerial parts of S. altissima (syn. S. canadensis) from Alabama were α-pinene (13.91%), sabinene (14.25%), myrcene (20.29%), bornyl acetate (14.44%), and germacrene D (10.67%; Table9). Previous examinations of S. canadensis essential oils have shown germacrene D to be one of the most abundant components (Table 10). However, Weyerstahl and co-workers [82] found curlone (23.5%) to be a major component of S. canadensis from Poland, Schmidt and co-workers [83] found cyclocolorenone (38%) to be a major component in S. canadensis from northern Germany, and Kasali and co-workers [84] found 6-epi-β-cubebene to be a major component (20.5%) in S. canadensis essential oil from Poland. Interestingly, none of these compounds was detected in the sample of S. altissima essential oil from Alabama.

Table 9. Chemical composition of the essential oil of Solidago altissima L.

RI a RI b Compound % SD RI a RI b Compound % SD ± ± 802 801 Hexanal 0.17 0.01 1382 1387 β-Bourbonene tr ± 850 846 (2E)-Hexenal 1.21 0.03 1386 1387 β-Cubebene 0.10 0.01 ± ± 921 921 Tricyclene 0.06 0.00 1387 1389 β-Elemene 0.18 0.00 ± ± 924 924 α-Thujene 1.30 0.00 1416 1419 β-Ylangene 0.14 0.01 ± ± 931 932 α-Pinene 13.91 0.04 1418 1417 β-Caryophyllene 0.50 0.03 ± ± 948 946 Camphene 2.41 0.01 1428 1430 β-Copaene 0.14 0.01 ± ± 971 969 Sabinene 14.25 0.03 1454 1452 α-Humulene 0.17 0.00 ± ± 976 974 β-Pinene 4.62 0.02 1473 1478 γ-Muurolene 0.59 0.03 ± ± 988 988 Myrcene 20.29 0.04 1477 1483 α-Amorphene 0.12 0.02 ± ± 1005 1004 (3Z)-Hexenyl acetate tr c 1479 1484 Germacrene D 10.67 0.03 ± 1006 1002 α-Phellandrene 2.84 0.02 1487 1489 β-Selinene tr ± 1016 1014 α-Terpinene 0.10 0.00 1490 1495 γ-Amorphene 0.59 0.01 ± ± 1024 1020 p-Cymene 2.26 0.00 1494 1500 Bicyclogermacrene 0.18 0.00 ± ± 1028 1024 Limonene 1.27 0.01 1496 1500 α-Muurolene 0.14 0.01 ± ± 1030 1025 β-Phellandrene 0.35 0.01 1511 1513 γ-Cadinene 0.30 0.01 ± ± 1044 1044 (E)-β-Ocimene 0.08 0.01 1513 1514 Cubebol 0.06 0.02 ± ± 1057 1054 γ-Terpinene 0.38 0.00 1516 1522 δ-Cadinene 0.68 0.02 ± ± 1069 1065 cis-Sabinene hydrate 0.24 0.00 1535 1537 α-Cadinene 0.09 0.01 ± ± 1084 1086 Terpinolene 0.15 0.01 1547 1548 Elemol 0.06 0.01 ± ± 1090 1090 6,7-Epoxymyrcene 0.05 0.00 1575 1574 Germacra-1(10),5-dien-4β-ol 0.14 0.01 ± ± 1099 1099 α-Pinene oxide tr 1581 1582 Caryophyllene oxide 0.06 0.01 ± 1101 1098 trans-Sabinene hydrate 0.15 0.00 1591 1594 Salvial-4(14)-en-1-one 0.06 0.01 ± ± 1105 1100 Nonanal tr 1619 1611 Germacra-1(10),5-dien-4α-ol 0.12 0.01 ± 1112 1113 (E)-4,8-Dimethylnona-1,3,7-triene 0.11 0.03 1627 1629 iso-Spathulenol 0.20 0.01 ± ± 1124 1124 cis-p-Menth-2-en-1-ol 0.05 0.00 1641 1638 τ-Cadinol 0.09 0.02 ± ± 1180 1174 Terpinen-4-ol 0.73 0.01 1643 1640 τ-Murrolol 0.14 0.01 ± ± 1195 1186 α-Terpineol 0.06 0.01 1645 1644 α-Muurolol (=δ-Cadinol) 0.10 0.01 ± ± 1203 1202 cis-Sabinol 0.50 0.01 1654 1652 α-Cadinol 0.41 0.03 ± ± 1219 1219 cis-Sabinene hydrate acetate 0.15 0.01 Green leaf volatiles 1.38 ± 1283 1287 Bornyl acetate 14.44 0.02 Monoterpene hydrocarbons 64.26 ± 1333 1335 δ-Elemene 0.05 0.00 Oxygenated monoterpenoids 16.37 ± 1345 1345 α-Cubebene 0.12 0.00 Sesquiterpene hydrocarbons 14.90 ± 1367 1373 α-Ylangene tr Oxygenated sesquiterpenoids 1.44 1368 1373 Linalyl isobutyrate tr Others 0.11 1374 1374 α-Copaene 0.05 0.00 Total Identified 98.45 ± a RI = Retention index determined in reference to a homologous series of n-alkanes on a ZB-5ms column. b RI values from the databases. c tr = “trace” (<0.05%).

Table 10. Comparison of the major components in Solidago altissima (syn. S. canadensis) essential oils.

Source of S. altissima (S. canadensis) Component Commercial Bimtal, Bimtal, Moscow, Giza, Slovakia Slovakia Hungary Poland Alabama (Young Living) India India Russia Egypt [88] [90] [91] [82] (This Work) [85] [86] [87] [89] [92] α-Pinene 13.3 5.0 0.4 1.8–36.3 28.1 11.6 4.6 29.2 14.7 13.9 Sabinene 8.0 2.4 0.3 — 0.5 3.9 0.1 — 0.2 14.2 β-Pinene — 1.2 0.2 0.5–6.5 2.8 3.1 1.2 4.8 1.5 4.6 Myrcene 6.3 2.8 — — 7.3 — tr 13.7 4.2 20.2 Limonene 11.0 12.5 4.2 4.3-9.0 7.0 12.5 1.0 9.6 9.3 1.3 Bornyl acetate 4.3 2.1 3.4 — 7.3 6.3 13.4 6.2 1.3 14.4 Germacrene D 34.4 56.7 64.1 0.0–11.1 39.2 34.9 11.0 10.3 19.8 10.6 Plants 2020, 9, 126 11 of 18

2.9. Xanthium strumarium L. The major volatile components from the aerial parts of X. strumarium were limonene (48.23%), myrcene (14.31%), germacrene D (13.92%), (2E)-hexenal (5.79%), and sabinene (4.89%; Table 11). The compositions of Xanthium strumarium essential oils from the Middle East have been reported, including Iran [93,94] and Pakistan [95]. The leaf essential oil from Khoramabad, Iran, was composed largely of limonene (24.7%), borneol (10.6%), bornyl acetate (5.9%), and β-cubebene (6.3%) [93]. The leaf essential oil from Zabol, Iran, was qualitatively similar, limonene (20.3%), borneol (11.6%), bornyl acetate (4.5%), and β-cubebene (3.8%), but also contained a large concentration of cis-β-guaiene (34.2%), which was not observed in any other X. strumarium essential oils [94]. The leaf essential oil of X. strumarium from Lahore, Pakistan, contained limonene (5.7%), β-caryophyllene (17.5%), spathulenol (6.1%), and α-cadinol (6.7%) as major components [95]. The differences in chemical compositions may be related to different genetic factors as well as geographical location; Tropicos® currently lists 13 subordinate taxa for X. strumarium [14].

Table 11. Chemical composition of the essential oil of Xanthium strumarium L.

RI a RI b Compound % SD RI a RI b Compound % SD ± ± 793 788 1-Octene 0.09 0.01 1417 1417 β-Caryophyllene 0.93 0.04 ± ± 801 797 (3Z)-Hexenal 0.11 0.01 1428 1430 β-Copaene 0.06 0.01 ± ± 802 801 Hexanal 0.75 0.07 1454 1452 α-Humulene 0.49 0.03 ± ± 859 846 (2E)-Hexenal 5.79 0.03 1479 1484 Germacrene D 13.92 0.05 ± ± 865 863 1-Hexanol 0.12 0.01 1487 1489 β-Selinene 0.11 0.01 ± ± 921 921 Tricyclene 0.05 0.01 1493 1500 Bicyclogermacrene 0.29 0.01 ± ± 924 924 α-Thujene 0.08 0.03 1496 1500 α-Muurolene 0.13 0.01 ± ± 931 932 α-Pinene 0.80 0.01 1511 1513 γ-Cadinene 0.19 0.01 ± ± 948 946 Camphene 0.95 0.02 1516 1522 δ-Cadinene 0.27 0.03 ± ± 971 969 Sabinene 4.89 0.02 1575 1547 Germacra-1(10),5-dien-4β-ol 0.21 0.01 ± ± 976 974 β-Pinene 0.30 0.01 1581 1582 Caryophyllene oxide 0.23 0.01 ± ± 978 974 1-Octen-3-ol 0.20 0.02 1641 1638 τ-Cadinol 0.31 0.02 ± ± 987 988 Myrcene 14.31 0.04 1643 1640 τ-Muurolol 0.23 0.02 ± ± 1004 1003 p-Mentha-1(7),8-diene 0.05 0.01 1654 1652 α-Cadinol 0.59 0.05 ± ± 1016 1014 α-Terpinene 0.06 0.01 1663 1668 ar-Turmerone 0.10 0.01 ± ± 1028 1024 Limonene 48.23 0.22 1693 1688 Shyobunol 0.27 0.03 ± ± 1030 1025 β-Phellandrene 0.90 0.03 1932 1931 Beyerene 0.64 0.02 ± ± 1044 1044 (E)-β-Ocimene 0.10 0.02 2105 2106 (E)-Phytol 0.25 0.03 ± ± 1057 1054 γ-Terpinene 0.16 0.01 Green leaf volatiles 6.77 ± 1069 1067 cis-Linalool oxide (furanoid) 0.06 0.01 Monoterpene hydrocarbons 70.87 ± 1099 1095 Linalool 1.16 0.01 Oxygenated monoterpenoids 2.22 ± 1180 1174 Terpinen-4-ol 0.39 0.01 Sesquiterpene hydrocarbons 16.58 ± 1219 1217 β-Cyclocitral 0.11 0.02 Oxygenated sesquiterpenoids 1.95 ± 1283 1287 Bornyl acetate 0.56 0.01 Diterpenoids 0.89 ± 1351 1356 Eugenol 0.28 0.03 Benzenoids 0.28 ± 1386 1387 β-Cubebene 0.10 0.02 Others 0.29 ± 1416 1419 β-Ylangene 0.08 0.03 Total Identified 99.85 ± a RI = Retention index determined in reference to a homologous series of n-alkanes on a ZB-5ms column. b RI values from the databases.

2.10. Antifungal Screening Depending on material available, the essential oils were screened for antifungal activity against the opportunistic fungal pathogens Aspergillus niger, Candida albicans, and Cryptococcus neoformans using the microbroth dilution technique (Table 12). The essential oil of E. serotinum showed promising antifungal activity against C. neoformans with a minimum inhibitory concentration (MIC) value of 78 µg/mL. The high concentration of cyclocolorenone in E. serotinum is likely responsible for the observed antifungal activity of this essential oil. Cyclocolorenone had been previously reported to show antifungal activity against Curvularia lunata, Chaetomium cochliodes, and Chaetomium spinusum [96]. Germacrene D may also contribute to the antifungal activity of E. serotinum essential oil as well as essential oils of E. macrophylla, P. canadensis, and R. laciniata. Germacrene D has shown antifungal activity against Aspergillus niger with MIC of 39 µg/mL [97]. Plants 2020, 9, 126 12 of 18

Table 12. Major components and antifungal activities of Asteraceae essential oils.

Antifungal Activity, MIC, µg/mL a Plant Species Major Components (>5%) in the Essential Oil Aspergillus Candida Cryptococcus niger albicans neoformans germacrene D (6.6%), palustrol (5.4%), Eupatorium serotinum Michx. 313 625 78 cyclocolorenone (23.5%) β-pinene (8.5%), limonene (28.6%), terpinolene Eurybia macrophylla (L.) Cass. 625 625 156 (5.3%), germacrene D (19.7%), germacrene B (7.0%) Eutrochium purpureum (L.) hexanal (6.8%), (2E)-hexenal (59.7%), methyl 625 625 625 E.E. Lamont salicylate (10.4%), eugenol (11.8%) α-pinene (19.6%), α-phellandrene (28.2%), Polymnia canadensis L. 625 625 156 germacrene D (11.4%) α-pinene (10.2%), β-pinene (9.2%), myrcene (5.3%), Rudbeckia laciniata L. 625 1250 156 limonene (58.9%) Silphium integrifolium Michx. α-pinene (58.5%), β-pinene (14.7%), myrcene (9.7%) n.t. b n.t. n.t. Smallanthus uvedalia (L.) α-pinene (62.3%), β-pinene (6.0%), limonene (11.3%) n.t. n.t. n.t. Mack. α-pinene (13.9%), sabinene (14.2%), myrcene (20.2%), Solidago altissima L. 625 1250 313 bornyl acetate (14.4%), germacrene D (10.6%) (2E)-hexenal (5.8%), myrcene (14.3%), limonene Xanthium strumarium L. 625 1250 n.t. (48.0%), germacrene D (13.9%) a Each minimum inhibitory concentration (MIC) determination was carried out in triplicate. b n.t. = not tested due to limited availability of the essential oil.

The modest antifungal activity of E. purpureum is somewhat surprising. The major components were hexanal, (2E)-hexenal, methyl salicylate, and eugenol. Hexanal [98] and (2E)-hexenal [99,100] are both known to be antifungal to plant pathogenic fungi. Methyl salicylate is only weakly antifungal against A. niger, C. albicans, or C. neoformans, but eugenol is somewhat active (see Table 13). Monoterpene hydrocarbons such as α-pinene, β-pinene, limonene, or myrcene show only weak antifungal activity (Table 13) and are not expected to contribute to the antifungal activities of the essential oils unless there are synergistic effects of these components (see, for example [101,102]). The mechanisms of antifungal activity of essential oils are poorly understood. However, it has been suggested that essential oils and their components, being lipophilic, can disrupt the membranes of fungi causing membrane permeability [103].

Table 13. Antifungal activities (MIC, µg/mL) of essential oil components.

Compound Aspergillus niger Candida albicans Cryptococcus neoformans α-Pinene 1250 625 313 β-Pinene 625 1250 625 Limonene 625 1250 625 Myrcene 625 625 625 Methyl salicylate 625 625 625 Eugenol 78 313 156 Bornyl acetate 625 625 625

3. Materials and Methods

3.1. Plant Material Aerial parts of each plant were collected from various sites in north Alabama (Table 14). Plants were identified by S.K. Lawson and voucher specimens were deposited in the University of Alabama in Huntsville herbarium (HALA). The fresh plant material (aerial parts) were chopped and hydrodistilled using a Likens–Nickerson apparatus with continuous extraction with CH2Cl2 for three hours. The solvent was evaporated to give pale yellow essential oils (Table 14). Plants 2020, 9, 126 13 of 18

Table 14. Plant collection sites and essential oil yields of Asteraceae from north Alabama.

Voucher Mass of Aerial Yield of Essential Plant Collection Site, Date Number Parts (g) Oil (mg) 34 38 29” N, 86 24 39” W, Eupatorium serotinum ◦ 0 ◦ 0 elev. 199 m 233754 49.09 6.4 (0.013%) Michx. 13 September 2018 34 39 25” N, 86 24 45” W, Eurybia macrophylla (L.) ◦ 0 ◦ 0 elev. 241 m 233117 56.56 10.6 (0.019%) Cass. 15 September 2018 34 38 40” N, 86 27 22” W, Eutrochium purpureum ◦ 0 ◦ 0 elev. 180 m 091843 63.44 12.3 (0.019%) (L.) E.E. Lamont 12 August 2018

34◦38029” N, 86◦24039” W, Polymnia canadensis L. elev. 199 m 184700 52.89 39.1 (0.074%) 21 July 2018

34◦42042” N, 86◦32038” W, Rudbeckia laciniata L. elev. 345 m 004426 54.53 6.0 (0.011%) 13 September 2018 34 42 42” N, 86 32 38” W, Silphium integrifolium ◦ 0 ◦ 0 elev. 345 m 004152 15.02 6.4 (0.043%) Michx. 15 September 2018 34 42 42” N, 86 32 38” W, Smallanthus uvedalia (L.) ◦ 0 ◦ 0 elev. 345 m 000714 56.21 5.9 (0.010%) Mack. 15 September 2018

34◦38040” N, 86◦27022” W, Solidago altissima L. elev. 180 m 001425 54.41 44.9 (0.083%) 12 August 2018

34◦38049” N, 86◦24038” W, Xanthium strumarium L. elev. 200 m 224724 69.69 7.0 (0.010%) 15 September 2018

3.2. Gas Chromatography–Mass Spectrometry The Asteraceae essential oils were analyzed by GC–MS using a Shimadzu GC–MS-QP2010 Ultra fitted with a Phenomenex ZB-5ms column as previously described [104]. Identification of the essential oil components was determined by comparison of their retention indices, determined with respect to a homologous series of n-alkanes and their mass spectral fragmentation patters with those from available databases (Adams [105], NIST17 [106], and FFNSC 3 [107]) or in our in-house library [108].

3.3. Gas Chromatography–Flame Ionization Detection Quantification of the essential oils was determined by GC–FID using a Shimadzu GC 2010 instrument fitted with a ZB-5 column [104], using the same parameters that were used for the GC–MS. The concentrations (average of three measurements standard deviations) are based on peak ± integration without standardization.

3.4. Antifungal Screening The essential oils were screened for antifungal activity against Aspergillus niger (ATCC 16888), Candida albicans (ATCC 18804), and Cryptococcus neoformans (ATCC 24607) using the microbroth dilution method as previously described [109]. Amphotericin B was used as the positive control and RPMI medium was used as the negative control. The antifungal assays were carried out in triplicate.

4. Conclusions There is much intraspecific variation in essential oil compositions of these members of the Asteraceae. Much of the variation can be attributed to geographical location or seasonal variation. Plants 2020, 9, 126 14 of 18

Eupatorium serotinum essential oil showed notable antifungal activity against Cryptococcus neoformans. However, the yield of this essential oil (0.013%) is too low to be considered as pharmacologically useful. If suitable sources of the major component cyclocolorenone can be identified, then this compound may serve as important antifungal template for further elaboration.

Author Contributions: Conceptualization, S.K.L. and W.N.S.; methodology, W.N.S., R.L.M., and P.S.; software, P.S.; validation, W.N.S.; formal analysis, W.N.S.; investigation, S.K.L., L.G.S., C.N.P., and P.S.; resources, R.L.M., P.S., and W.N.S.; data curation, W.N.S.; writing—original draft preparation, W.N.S.; writing—review and editing, S.K.L., L.G.S., C.N.P., R.L.M., P.S., and W.N.S.; supervision, W.N.S. and R.L.M.; project administration, W.N.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: P.S. and W.N.S. participated in the project as part of the activities of the Aromatic Plant Research Center (APRC, https://aromaticplant.org/). Conflicts of Interest: The authors declare no conflict of interest.

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